Various optical sectioning techniques have provided pathologists and clinicians with some ability to image biological samples noninvasively at or below a surface of a target specimen, such as the skin of a person. Existing methods for optical sectioning include confocal microscopy, which is the most common method. Confocal microscopy works by passing received light through a pinhole, which rejects out-of-focus light from reaching a detector. Another optical sectioning method, known as structured illumination microscopy (SIM), does not require the use of a pinhole. SIM has an advantage over confocal microscopy because SIM can use wide field imaging and eliminate the need to raster scan.
In SIM, a high-frequency pattern may be used to modulate a plane of interest. Decoupling of in-focus light from out-of-focus light for a given image is achieved by phase shifting the modulation pattern to at least three different positions and then pair-wise subtracting them from one another. Accordingly, three different images are sequentially acquired at different modulation pattern phases to be able to achieve decoupling for a given image produced by SIM. Traditional SIM has also been extended to decouple in-focus and out-of-focus light based on acquisition of two different images.